The long-term goal of this project is to define the role of the novel lipid pathway mediated by sphingosine-1- phosphate (S1P) in inflammation and to establish components of this pathway as potential novel targets for anti-inflammatory therapy. The PI's laboratory has an established track record of expertise in sphingolipid metabolism and function. Studies from the previous funding period have led us into a novel exciting direction on the role and regulation of S1P in inflammation. S1P is generated by phosphorylation by sphingosine kinase (SK) in many cell types including inflammatory cells. S1P acts extracellularly by binding to members of the G-protein coupled receptors S1P1-5, or intracellularly on poorly defined targets. It mediates several biologic activities, including mitogenesis, cell survival, angiogenesis and inflammatory responses. In this competing renewal we have compelling new data, whereby we have implicated this pathway as a key regulator of cyclooxygenase-2 (COX-2) expression and prostaglandin E2 (PGE2) production. In addition we find that SK1 is significantly overexpressed in inflammatory tissues. We also demonstrate that bacterial lipopolysaccharide (LPS) regulates SK1. This proposal will therefore, test the hypothesis that SK1 and S1P regulate inflammation, and that inhibiting this pathway could inhibit inflammatory responses. To test this hypothesis we propose the following aims: 1) Establish that the SK1 and S1P pathway is regulated in inflammation and determine the mechanisms of this regulation. This will be done by determining if this pathway is regulated in inflammation and studying the mechanisms of this regulation in cell models of inflammation, and by determining the expression and cell-type distribution of SK1 in inflammatory tissues from humans and from animal models of inflammation. 2) Establish the function of SK1 and S1P in regulation of inflammation and determine the mechanisms of action of this pathway. This will be done by demonstrating that SK1 and S1P have a significant role in regulating inflammation by evaluating the effect of over expression of SK1 and of S1P treatment in cells and in vivo models of inflammation, and determining the mechanisms by which SK1 and S1P regulate inflammatory pathways (NF-KB, IL2, TNF, ERKs, COX-2). 3) Determine if SK1 and S1P are necessary for inflammation. This will be done by blocking SK1 activity in cells and in vivo using small interfering RNA to SK1 and/or dominant negative SK1. In addition we will test different compounds that we synthesized for their ability to inhibit SK1 or to antagonize S1P receptors in cells and in vivo models of inflammation. These studies will enable us to gain important insight into the role of this pathway in inflammatory responses and may also provide novel therapeutic approaches to inflammation.